Inosine-5′-monophosphate dehydrogenase (IMPDH; EC 1.1.1.205) is a key enzyme in the de novo synthesis of purines. IMPDH catalyzes the NAD-dependent oxidation of inosine-5′-monophosphate (IMP) to xanthosine-5′-monophosphate (XMP), resulting in the production of NADH and XMP. IMPDH catalyzed oxidation of IMP to XMP is the rate-limiting step in the synthesis of guanine nucleotides.
IMPDH exists as a homotetramer (i.e., the enzyme has four subunits each comprising an IMPDH polypeptide). In many species, two isoforms of IMPDH have been described and are designated type I and type II IMPDH. Human type I and type II IMPDH have been identified and sequenced. Human IMPDH types I and II are both 514 amino acids in length and they share 84% sequence identity. The nucleotide and amino acid sequence of human type I IMPDH are disclosed in Natsumeda, Y., et al., J. Biol. Chem. 265:5292-5295 (1990), and the nucleotide and amino acid sequence of human type II IMPDH are disclosed in Natsumeda, Y., et al., J. Biol. Chem. 265:5292-5295 (1990), Collart, F. R. and Hubermann, E., J. Biol. Chem. 263:15769-15772 (1988), and U.S. Pat. No. 5,665,583. The subunits of human IMPDH types I and II that make up the IMPDH homotetramer each have a subunit molecular weight of 56 kDa. Human type II IMPDH has a catalytic core domain (amino acids 1-109 and 245-514) and a subdomain (amino acids 110-244) with unknown function.
IMPDH is a target for antitumor (e.g., antileukemic) therapy and immunosuppressive chemotherapy. IMPDH is upregulated in neoplastic and differentiating cells. Furthermore, proliferating B and T lymphocytes depend on the de novo pathway, rather than the salvage pathway, for synthesis of guanine nucleotides with inhibition of guanine nucleotide synthesis resulting in inhibition of DNA synthesis. Thus, IMPDH is an important enzyme for B and T cell proliferation, and inhibition of IMPDH activity inhibits both B and T cell proliferation making IMPDH an important target for immunosuppressive chemotherapy.
Mycophenolic acid (MPA) is an uncompetitive inhibitor of human types I and II IMPDH and MPA binds IMPDH after NADH is released, but before XMP is produced. MPA is the active metabolite in vivo of the ester prodrug mycophenolate mofetil (CellCept; MMF). MMF is an immunosuppressant that blocks B and T cell proliferation and MMF has been approved for the treatment of kidney and heart transplant rejection. MMF has also been used clinically to treat cancer and viral infections, and has been used as an anti-vascular hyperproliferative agent, an antipsoriatric agent, an antibacterial agent, an antifungal agent, and has been used for the treatment of autoimmune diseases.
MMF is hydrolyzed to MPA in vivo, and, accordingly, the monitoring of MPA levels in vivo allows for monitoring of MMF dosages. The measurement of MPA levels in patients treated with MMF is of clinical significance because the monitoring of MPA levels improves therapeutic efficacy, e.g., optimal MMF levels necessary for adequate immunosuppression can be determined, and minimizes the adverse side effects of the drug. Isolated, recombinant IMPDH has been used in assays to measure MPA levels in patients treated with MMF, such as the assays described in U.S. Pat. Nos. 6,107,052 and 6,524,808.
The ability to produce and to isolate large amounts of stable, recombinant IMPDH (e.g., IMPDH that aggregates minimally) is important for use in assays for monitoring MPA levels in patients treated with MMF, or for use in assays to monitor the levels in patient samples of any other therapeutically useful IMPDH inhibitor. Other inhibitors of IMPDH are described in Anderson, J. H. et al., J. Biol. Chem. 243:4762-4768 (1968) and in U.S. Pat. Nos. 5,380,879, 5,444,072, and 5,807,876 and in PCT publications WO 94/01105 and WO 94/12184.
The ability to produce and to isolate large amounts of stable, recombinant IMPDH is also important for other clinical and research applications, such as for the identification and design of new IMPDH inhibitors useful for cancer and immunosuppressive therapies, and for determining the sensitivity of IMPDH to those inhibitors.
The present invention is directed to modified recombinant IMPDH polypeptides, and to isolated, modified nucleic acid molecules encoding these modified recombinant IMPDH polypeptides. The invention is also directed to a method, vectors, and host cells for producing such a modified recombinant IMPDH polypeptide, and to kits comprising the modified IMPDH polypeptide. The recombinant IMPDH polypeptides are modified to contain a histidine tag for purification by nickel chelate affinity chromatography, and are also modified in the subdomain region of the polypeptides so that the rate stability of the histidine-tagged, modified IMPDH polypeptides is maintained relative to the wild-type IMPDH polypeptide.
In one embodiment, the histidine-tagged, modified IMPDH polypeptide has the amino acid sequence as shown in any one of SEQ ID NOS: 14, 16, 18, 20, or 22. In another embodiment, one or more of the amino acids at positions 116-250 as shown in SEQ ID NO: 12 are substituted in the histidine-tagged, modified IMPDH polypeptide with a negatively charged amino acid. In still another embodiment, one or more of the positively charged amino acids at positions 130, 132, 134, 140, 142, 143, 155, 159, 167, 173, 177, 187, 188, 201, 209, 211, 212, 214, 230, 234, 235, 237, 244, 247, or 248 as shown in SEQ ID NO: 12 are substituted in the histidine-tagged, modified IMPDH polypeptide with a negatively charged amino acid. In an alternative embodiment, one or more of the positively charged amino acids at positions 130, 132, 134, 140, 142, 155, 159, 167, or 173 as shown in SEQ ID NO: 12 are substituted in the histidine-tagged, modified IMPDH polypeptide with a negatively charged amino acid.
Also provided are isolated nucleic acid molecules encoding the histidine-tagged, modified IMPDH polypeptides. Accordingly, the isolated nucleic acid molecules provided herein encode a modified type I or type II IMPDH polypeptide wherein the modified IMPDH polypeptide has a histidine tag and wherein the subdomain of the IMPDH polypeptide is modified so that the rate stability of the histidine-tagged, modified IMPDH polypeptide is maintained relative to the wild-type IMPDH polypeptide. In one embodiment, the isolated nucleic acid molecule can comprise a sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, and complementary sequences thereof. In another embodiment, isolated nucleic acid molecules are provided wherein the complementary sequences of the isolated nucleic acid molecules hybridize under stringent conditions to any one of the nucleotide sequences set forth in SEQ ID NO: 13, 15, 17, 19, or 21.
In still another embodiment, isolated nucleic acid molecules are provided wherein the complementary sequences of the isolated nucleic acid molecules hybridize under stringent conditions to nucleotides 346-750 of a sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID NO: 21. In still another embodiment, isolated nucleic acid molecules are provided comprising nucleotides 346-750 of a sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID NO: 21. In another embodiment, isolated nucleic acid molecules are provided wherein the IMPDH polypeptide encoded comprises an amino sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, and SEQ ID NO: 22. In still another embodiment, a vector is provided comprising the isolated nucleic acid molecule of SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, or SEQ ID NO: 21 obtainable from E. coli H712 and having ATCC accession number PTA-5786, PTA-5782, PTA-5784, PTA-5785, and PTA-5783, respectively.